Efficacy of nitrous oxide in adults undergoing puncture biopsy: A systematic review and meta-analysis of randomized controlled trials

Background Nitrous oxide (N2O) with rapid analgesic effect is often used to relieve pain induced by diagnostic procedures. This review was conducted to evaluate the efficacy and safety of N2O in patients undergoing puncture biopsy. Methods We systematically searched PubMed, Embase, the Cochrane Library, Web of Science, Scopus and the ClinicalTrials.gov up to March, 2022. Randomized controlled trials (RCTs) were included if they investigated the effect of N2O in adults undergoing puncture biopsy. The primary outcome was pain score. Secondary outcomes included anxiety score, patient satisfaction and side effects. Results Twelve RCTs with 1070 patients were included in the qualitative review, of which eleven RCTs were included in the meta-analysis. Pooled analysis suggested that compared with the controls (placebo, lidocaine and midazolam), N2O had better analgesic effect (MD -1.12, 95% CI -2.12 to -0.13, P = 0.03; I2 = 94%). In addition, N2O significantly alleviated patient anxiety (MD = -1.79, 95% CI -2.41 to -1.18, P<0.00001; I2 = 0%) and improved patient satisfaction (MD 1.81, 95% CI 0.11 to 3.50, P = 0.04; I2 = 92%). There was no significant difference regrading the risk of nausea (RR 2.56; 95% CI 0.70 to 9.31, P = 0.15; I2 = 0%), headache (RR 0.62, 95% CI 0.17 to 2.33, P = 0.48; I2 = 46%), dizziness (RR 1.80, 95% CI 0.63 to 5.13, P = 0.27; I2 = 0%) or euphoria (RR 2.67, 95% CI 0.81 to 8.79, P = 0.11; I2 = 8%) between the N2O group and the control group. Conclusion The present review suggested that N2O might be effective for pain management in patients undergoing puncture biopsy.


Introduction
Puncture biopsy is the main way to gain tumor tissue or cell sample for histopathological diagnosis [1], such as percutaneous liver biopsy (PLB), lumbar puncture (LP), bone marrow puncture and transrectal ultrasound-guided prostate biopsy (TUSPB). It can provide strong proof for identification of benign and malignant tumors and selection of the appropriate remedy scheme [2][3][4][5][6]. Puncture biopsy is painful and unpleasant [7][8][9][10], and the anxiety and fear also increase the perception of pain, which may hinder the patient compliance with future biopsy.
Nitrous oxide (N 2 O) is an inhalational agent with analgesic, sedative and anti-anxiety properties [11]. It has the advantages of simple handling, fast onset and few adverse reactions and has been shown to have good analgesic effects in various settings such as dental, emergency treatment, obstetrics and pediatrics [12][13][14][15][16]. Recently, several randomized controlled trials (RCTs) investigated the effects of N 2 O regarding pain management in patients undergoing puncture biopsy and no consistent conclusion was made. Thus, we conducted a systematic review and meta-analysis of RCTs to assess the efficacy and safety of N 2 O in patients undergoing puncture biopsy.

Materials and methods
This systematic review and meta-analysis was conducted following the current recommendations of the Cochrane Collaboration and was reported according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses [17]. The protocol was registered on PROSPERO (registration number: CRD42022321185).

Literature search and eligibility criteria
Relevant articles were identified by searching PubMed, Embase, the Cochrane Library, Web of Science, Scopus and the ClinicalTrials.gov (up to March, 2022) without language restriction. Electronic searches were conducted using the Exploded Medical Subject Headings and appropriate corresponding keywords: "nitrous oxide", "Entonox", "N 2 O", "puncture", ''biopsy" and ''puncture biopsy". We also inspected the reference lists of studies identified by the previous searches for additional studies eligible for inclusion. Two authors independently assessed the eligibility of all studies identified in the initial research. Studies meeting the following criteria were included: (1) Population: adults undergoing puncture biopsy; (2) Intervention: N 2 O inhalation; (3) Comparison: placebo or other analgesic methods; (4) Outcome: at least one of the following endpoints: pain score, anxiety score, patient satisfaction and side effects; (5) Design: randomized controlled trial.

Data extraction
Two authors independently extracted the following data: first author, year of publication, country, number of patients, patient characteristics, interventions, controls, adjuvant analgesics and main outcomes. We would contact the original authors by e-mail if data needed clarification or were not presented in the article. Extracted data were checked by the third author and any dispute was settled by consultation.
The primary outcome was pain score. Secondary outcomes included anxiety score, patient satisfaction and side effects. The pain score, anxiety score, and patient satisfaction were expressed using a visual analogue scale (VAS) or numeral rating scale (NRS) and was transformed to a 0-10 cm scale when the data were reported on a 0-100 mm scale. The data on the side effects were dichotomous. If the outcome data were not reported by mean±SD, they were converted according to the Cochran Handbook [18]. The definition of each outcome mentioned above was the same as that used in each included trial.

Risk of bias and evidence grade assessment
Two authors independently assessed risk of bias in the included RCTs with the method recommended by the Cochrane Collaboration [19]. The quality of evidence for the outcomes were appraised using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach [20]. Divergences were solved by discussion.

Statistical analysis
The statistical analysis was conducted using Review Manager software (version 5.3; Nordic Cochrane Centre, Cochrane Collaboration). Differences were shown as the risk ratio (RR) with 95% confidence interval (CI) for dichotomous outcomes, and the weighted mean difference (MD) with 95% CI for continuous outcomes. The heterogeneity between the studies was assessed using I 2 . If I 2 was<50%, the studies were considered as homogeneous, and a fixedeffect model was used. If I 2 was �50%, there was significant heterogeneity among the studies, and a random-effects model was used. Potential sources of heterogeneity were identified by sensitivity analysis conducted by omitting one study in each turn and investigating the influence of a single study on the overall pooled estimate. Prior subgroup analyses were conducted to explore the influence of type of puncture biopsy, different controls, and gender on the overall pooled estimate. Publication bias was appraised by visually examining a funnel plot. P < 0.05 was considered statistically significant.

Results
The overall search yielded 2229 citations, of which 2199 were weeded out for various reasons based on the title and abstract. The full texts of the remaining 30 publications were scrutinized for further assessment and then twelve RCTs [21][22][23][24][25][26][27][28][29][30][31][32] were eligible for qualitative systematic review. One study [31] reported the results of the questionnaires completed by the physicians and patients regarding their degree of satisfaction with the examination process on a visual analog scale score, which was not consistent and could not been combined with the results of the other included RCTs. One study [32] reported the pain score as median and range and the data was converted as mean and SD in the pooled analysis [18,33,34]. Finally, eleven RCTs [21][22][23][24][25][26][27][28][29][30]32] were included for meta-analysis (Fig 1).

Study characteristics
The included RCTs were published between 2001 and 2022 with sample sizes ranging from 41 to 183. Among the included trials, 542 underwent experimental arm and 528 patients were in the control arm. Among the selected trials, ten [21-27, 29, 30, 32] were conducted in Europe, one [31] in Asia, and one [28] in South America. The participants in one trial [30] were only female, three [22,23,28] were only male, and the remaining eight [21, 24-27, 29, 31, 32] contained males and females. Two trials included three groups, of which one trial [22] included N 2 O group, air group and blank group, and the other trial [23]

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ultrasound-guided fine needle aspiration for digestive tract diseases [31]. The characteristics and outcomes of included trials were presented in Table 1.

Quality of evidence and publication bias
The quality of evidence was low for pain score and moderate for the other remaining outcomes, which was shown in Table 2. As for publication bias of the pain score of N 2 O compared with the control group, visually inspecting funnel plots showed no evidence of potential publication bias among the included RCTs [35]

Discussion
To our best knowledge, this was the first systematic review and meta-analysis on the efficacy and safety of N 2 O in adults undergoing puncture biopsy. The accumulated evidence from the present study showed that N 2 O might be an effective way of analgesia and sedation, which relieved pain perception and anxiety in patients undergoing diagnostic painful procedures. Additionally, compared with other analgesic methods, N 2 O had no excessive side effects.
To date, puncture biopsy is still a crucial method for the identification and analysis of pathological tissue and cell morphology to help doctors to make the pathological diagnoses. Generally speaking, conventional puncture biopsy is conducted when the patient is conscious. Pain, fear and discomfort with regard to the puncture biopsy often reduce patient's compliance, which may delay the timely diagnosis and treatment of the tumor. Nowadays, a common analgesia mode is local anesthesia with lidocaine [36] in minor operations or invasive procedures like biopsies and minor excisions [37,38]. However, many patients complain that local anesthesia does not provide enough analgesia during puncture biopsy. Eisenberg et al. [39] reported that 69% of patients felt pain immediately after the operation, and more than half of patients were still in pain four hours later. Several studies also suggested that local anesthesia fail to provide sufficient early and late analgesia [7][8][9][10]. Although Chakupurakal et al's study [32] indicated that midazolam was superior to N 2 O in providing pain relief during bone marrow aspirate and trephine biopsy, midazolam caused respiratory depression, and care must be taken to monitor respiratory function which may prolong the length of hospital stay. Our pooled analysis indicated that compared with the controls, N 2 O had better analgesic effect with significant heterogeneity. The heterogeneity could be caused by several critical factors, such as the gender of patients, type of operation, control group, ratio of N 2 O/O 2 , inhalation time, the time point of data collection, and the number of puncture biopsies. Considering the limited number of studies included, the findings in the subgroup analyses should be evaluated rigorously and more high-quality RCTs are needed in the future. N 2 O was a self-administered inhaled gas reserved in a pre-prepared cylinder and trained nurses can conduct without the presence of professional anesthesiologists. Its action is rapid and reversible after stopping inhalation and N 2 O has no depressive effects on the respiratory or cardiovascular function [40], and does not obscure the signs and symptoms that may be necessary for disease diagnosis. The mixture of 20% N 2 O and 80% oxygen has an equivalent

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analgesic effect as well as a good sedative effect with 15 mg morphine [41]. Three trials [27,29,30] reported the anxiety score. Pooled results suggested that N 2 O significantly reduced patients' anxiety. Notably, Clark et al. [42] reported that N 2 O has amnesia characteristics so that patients often express an inability to recall severe pain or tension, even the entire procedure. In recent years, several studies have shown that N 2 O can be used in minor dental surgery, labor analgesia, pediatric operation pain (such as botulinum toxin injections, vaccine administration and intravenous catheter placement), and trauma first aid [43][44][45][46]. As for nausea, headache, dizziness and euphoria, pooled analysis showed that there was no significant difference between the N 2 O group and the control group. This finding may be related to the short inhalation time or low concentration of N 2 O. Most of the included studies applied 50% N 2 O/O 2 inhalation 5 min before and during the biopsy, and the N 2 O/O 2 inhalation was stopped at the end of the operation. In addditon, the frequency of side effects was relatively low among the included studies. Other side effects recorded in the included studies included oxygen desaturation (pulse oximetric saturation �94%), arterial hypotension, bradycardia, numbness and drowsiness, which usually disappeared rapidly after discontinuation [47]. N 2 O is a greenhouse gas and anesthesia is responsible for approximately 1% of N 2 O emissions in the atmosphere. Hence, N 2 O should be used by trained people and attention should be paid to scavenging it properly.
There were several limitations in the present study. Firstly, several included RCTs did not describe randomization and blinding in detail and the included trials were mostly singleblinded, which might affect the reliability of the pooled results. Secondly, the type of puncture biopsy and control groups varied among the included RCTs, which might lead to observed heterogeneity and thus impairing the robustness of our findings. Thirdly, several secondary outcomes and subgroup analysis only included not more than two RCTs, which need more large sample RCTs in the future so as to increase the reliability of the results. Fourthly, the outcome data in several included RCTs were not expressed as mean±SD. The measurement tools and time point of data collection also varied in the included studies. The data conversion and standardization were conducted in the pooled analysis, which might impair the robustness of  the results. Finally, pain and anxiety perception were subjective outcomes and were not likely to be consistently assessed by individual patients.

Conclusion
In summary, the present systematic review and meta-analysis suggested that the use of N 2 O might provide a better analgesic effect and slight side effects for puncture biopsy. However, due to the poor overall quality of the included studies and limited evidence, more welldesigned randomized controlled trials are still needed to confirm our findings in the future.